VP16 is a herpes simplex virus protein that functions as a potent transcriptional activator of the viral immediate early genes. The transcriptional activation domain of VP16 has been widely adopted in studies of the mechanisms of transcriptional activation in eukaryotes. The overall goal of this project is to understand the structure of the VP16 activation domain, and the nature of its interactions with the host cell transcriptional machinery. VP16 is known to be a phosphoprotein when present in the virion and in infected cells; moreover, the Gal4-VP16 fusion protein frequently used in mechanistic studies is phosphorylated as a consequence of transcriptional activation. We have used MALDI-MS and peptide mass mapping as one tool in our approach to defining the phosphorylation of VP16 and of Gal4-VP16. We aim to identify the amino acid(s) that are modified, and any changes in phosphorylation that occur during the infectious cycle; mass spectrometry provides a sensitive and accurate assay.